Genome-Scale Mining of Acetogens of the Genus Clostridium Unveils Distinctive Traits in [FeFe]- and [NiFe]-Hydrogenase Content and Maturation

ABSTRACT Knowledge of the organizational and functional properties of hydrogen metabolism is pivotal to the construction of a framework supportive of a hydrogen-fueled low-carbon economy. Hydrogen metabolism relies on the mechanism of action of hydrogenases. In this study, we investigated the genomes of several industrially relevant acetogens of the genus Clostridium (C. autoethanogenum, C. ljungdahlii, C. carboxidivorans, C. drakei, C. scatologenes, C. coskatii, C. ragsdalei, C. sp. AWRP) to systematically identify their intriguingly diversified hydrogenases’ repertoire. An entirely computational annotation pipeline unveiled common and strain-specific traits in the functional content of [NiFe]- and [FeFe]-hydrogenases. Hydrogenases were identified and categorized into functionally distinct classes by the combination of sequence homology, with respect to a database of curated nonredundant hydrogenases, with the analysis of sequence patterns characteristic of the mode of action of [FeFe]- and [NiFe]-hydrogenases. The inspection of the genes in the neighborhood of the catalytic subunits unveiled a wide agreement between their genomic arrangement and the gene organization templates previously developed for the predicted hydrogenase classes. Subunits’ characterization of the identified hydrogenases allowed us to glean some insights on the redox cofactor-binding determinants in the diaphorase subunits of the electron-bifurcating [FeFe]-hydrogenases. Finally, the reliability of the inferred hydrogenases was corroborated by the punctual analysis of the maturation proteins necessary for the biosynthesis of [NiFe]- and [FeFe]-hydrogenases. IMPORTANCE Mastering hydrogen metabolism can support a sustainable carbon-neutral economy. Of the many microorganisms metabolizing hydrogen, acetogens of the genus Clostridium are appealing, with some of them already in usage as industrial workhorses. Having provided detailed information on the hydrogenase content of an unprecedented number of clostridial acetogens at the gene level, our study represents a valuable knowledge base to deepen our understanding of hydrogenases’ functional specificity and/or redundancy and to develop a large array of biotechnological processes. We also believe our study could serve as a basis for future strain-engineering approaches, acting at the hydrogenases’ level or at the level of their maturation proteins. On the other side, the wealth of functional elements discussed in relation to the identified hydrogenases is worthy of further investigation by biochemical and structural studies to ultimately lead to the usage of these enzymes as valuable catalysts.

in several cases. For the electron-bifurcating type hydrogenases identified, comparison to the diaphorase subunit of complex 1 identified several differences in structural motifs hypothesize to be relevant to NAD(P)H cofactor binding.
Overall, I found that the manuscript fills an important knowledge gap in hydrogenase literature as hydrogenases from clostridial acetogens are less studied. The manuscript is well referenced and provides a helpful interpretation of the genomic results considering previous experimental work and provides basis for future characterization of the hydrogenases identified. Several aspects of the manuscript could be improved to increase accessibility to the general audience and to strengthen the impact of the work. Notably missing is a conclusions section, which should be added to summarize the primary findings and relevance to the field.
Specific comments: The title doesn't adequately reflect the content, as the title is specific for [FeFe]-hydrogenase but the work informs on [NiFe]-and [FeFe]-hydrogenase. For the introduction (paragraph starting on line 83), it would be helpful to incorporate a figure depicting relevant acetogen energetic pathways with proposed functional roles of [FeFe]-and [NiFe]-hydrogenases. On lines 78-80, the statement that "H2 evolution is carried out by [FeFe]-or [NiFe]-hydrogenases while H2 oxidation is typically but not exclusively, attributed to [NiFe]-hydrogenases" is somewhat confusing as there are many examples of [FeFe]-hydrogenases that show preference for H2 oxidation over H2 evolution. Line 83, there is a typo in "through." Figure legends should be added for Figures 1 and 2. I found Figure 1 a bit hard to follow with the different abbreviations and these could be spelled out in the legend. In the results section starting on line 123, "groups" of hydrogenases are referred to, however the terminology is not properly introduced. I gather this is taking on the classification system by Calusinska et al in Microbiology 2010, 156, 1575-1588 A few sentences about how [NiFe]-and [FeFe]-are classified into different groups and what these are would be helpful before reporting the results. Line 176 states that no representative [FeFe]-hydrogenase of subgroup B has been biochemical characterized. It should be noted that in Artz et al., J. Am. Chem. Soc. 2020, 142, 1227 there is initial biochemical and spectroscopic characterization of CpIII [FeFe]-hydrogenase from C. pasterianum (group B2 as reported by Therien et al. Frontiers Microbiology, 2017, 1305 which show an altered activity profile and spectroscopic properties compared to CpI (Group A2) and CpII (Group A3) [FeFe]-hydrogenases. For the paragraph starting on Line 178, a figure or schematic depicting how electron-bifurcating (or confurcating) [FeFe]-hydrogenase function in redox pathways via H2 uptake or evolution directions would be helpful. Line 456 (and 636), would recommend modifying the title "Redox cofactor binding subunits" to something that includes diaphorase since redox cofactor could be interpreted as something else. For Figures 7 and 8, would recommend incorporating a structural depiction of NuoF showing cofactor binding regions and conserved loops, using the coloring scheme of sequence alignment. The structural results with I-Tasser should also be brought into Figure 7 so any differences in the cofactor binding region of the hydrogenase diaphorase compared to NuoF can be visualized.
Reviewer #2 (Comments for the Author): Dear authors, I found your manuscript on hydrogenases rather intriguing because, as you mentioned in your introduction, they are widespread and fulfill diverse functions in microorganisms. Unfortunately I found the manuscript rather challenging to read, as there is no clear flow of thought throughout the manuscript. Much of the results and discussion part reads more like a review article, and it is difficult to identify what role your own results play. It reads more like a list of all the facts that you could find in the literature than a well conducted discussion of your own results. This makes your results and discussion part way too long and you lose the reader along the way. Looking at your data in the tables and figures, you seem to have down some quite solid work, so you should really try to emphasize what you have done and only represent that part of the literature that is clearly needed in the discussion of your data. I would suggest that you separate the "results and discussion" into two separate chapters, "results" and "discussion", in order to better focus on your own results.

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We provide a point-by-point responses to the issues raised by the Reviewers. The comments of the Reviewers are in blue, the responses of the Authors are reported in black and the changes applied to the manuscript (when transferrable directly in the present document) are reported in red.

Reviewer #1 (Comments for the Author):
The manuscript by Di Leonardo et al provides an in-depth genomic analysis of clostridial acetogen type [FeFe]-and [NiFe]-hydrogenases. The approach was to identify hydrogenases from select acetogen genomes and validate via a computational workflow which involved comparison to a database of hydrogenase sequences, with further validation provided by reference to known catalytic sequence motifs and analysis of neighboring genes. The results were further analyzed in context of their maturation genes required for biosynthesis, which revealed several interesting insights regarding absences of conserved genes in several cases. For the electron-bifurcating type hydrogenases identified, comparison to the diaphorase subunit of complex 1 identified several differences in structural motifs hypothesize to be relevant to NAD(P)H cofactor binding.
Overall, I found that the manuscript fills an important knowledge gap in hydrogenase literature as hydrogenases from clostridial acetogens are less studied. The manuscript is well referenced and provides a helpful interpretation of the genomic results considering previous experimental work and provides basis for future characterization of the hydrogenases identified. Several aspects of the manuscript could be improved to increase accessibility to the general audience and to strengthen the impact of the work. Notably missing is a conclusions section, which should be added to summarize the primary findings and relevance to the field.
We thank the Reviewer for his/her comments. Even though we did not created a dedicated Conclusion section, we restructured the manuscript to enhance the ability to discern our findings from the context of the literature that was used to interpret our results, in line with the request formulated by the Reviewer #2. Within this attempt, we separated the Results section from the Discussion one and, at the end of the latter, we included a brief summary of our results. We modified the manuscript as follows: We thank the Reviewer for asking for this supplement of information that we integrated in the  (B) With CO as electron source, the reducing equivalents for the reductive steps are provided by the CO dehydrogenase/acetyl coenzyme A synthase (CODH/ACS) which reduces Fd. The hydrogenase protects the cells from over-reduction when NADP and ferredoxin get too reduced during growth on CO.
The electron-bifurcating and ferredoxin-dependent transhydrogenase Nfn is transferring electrons between Fd, NADH and NADPH. The methylene-THF reductase is assumed to be electron bifurcating. Excess reduced ferredoxin (Fdred) is oxidized by the Rnf complex, which reduces NAD and builds up an H + gradient. We thank the Reviewer for pointing out this confusing sentence that we removed from the revised version of the manuscript.
Line 83, there is a typo in "through." We amended the manuscript correspondingly.     In the results section starting on line 123, "groups" of hydrogenases are referred to, however the terminology is not properly introduced. I gather this is taking on the classification system by Calusinska et al in Microbiology 2010Microbiology , 156, 1575Microbiology -1588  We thank the Reviewer for pointing out this aspect. We amended it by introducing the classification scheme in the Results section (lines 116-125 of the compare copy of the manuscript) as follows: "To this aim, we relied on the hydrogenase classification scheme predictive of biological functionality available at HydDB (20), which descends from the definition provided in refs. (1,18,21,22 We thank the Reviewer for noting this mistake. The revised version of the manuscript now includes this information in the Results section "[FeFe]-hydrogenases in clostridial acetogens" (lines 188-194 of the compare copy of the manuscript). Indeed, the Results section of the manuscript was modified as follows: "A representative hydrogenase of this subgroup, CpIII in C. pasteurianum, has been recently biochemically and spectroscopically characterized in comparison to the group A2 CpI and to the group A3 CpII 36). Preferential stabilization of key catalytic intermediates through subtle changes in the outer coordination sphere was found to result in stabilization and/or destabilization of different oxidation states. As a result of this characterization, CpIII was found to have a catalytic bias towards H2 production (35,36)." The Discussion section was modified as follows:

More recently, a biochemical and spectroscopic characterization of the [FeFe]-
hydrogenase CpIII from C. pasterianum, which belongs to group B2 as reported in (36)  Line 456 (and 636), would recommend modifying the title "Redox cofactor binding subunits" to something that includes diaphorase since redox cofactor could be interpreted as something else.
We thank the Reviewer for this suggestion and we adopted the following title: "Diaphorase subunits in electron-bifurcating [FeFe]-hydrogenases".

Reviewer #2 (Comments for the Author):
Dear authors, I found your manuscript on hydrogenases rather intriguing because, as you mentioned in your introduction, they are widespread and fulfill diverse functions in microorganisms.
Unfortunately I found the manuscript rather challenging to read, as there is no clear flow of thought throughout the manuscript. Much of the results and discussion part reads more like a review article, and it is difficult to identify what role your own results play. It reads more like a list of all the facts that you could find in the literature than a well conducted discussion of your own results. This makes your results and discussion part way too long and you lose the reader along the way. Looking at your data in the tables and figures, you seem to have down some quite solid work, so you should really try to emphasize what you have done and only represent that part of the literature that is clearly needed in the discussion of your data. I would suggest that you separate the "results and discussion" into two separate chapters, "results" and "discussion", in order to better focus on your own results.
We thank the Reviewer for his/her comments. In line with the suggestions, we restructured the manuscript in order to highlight the findings of our work. To this aim, we created separated sections for presenting the results and for their discussion in the context of the literature. We belive that, thanks to this change, the manuscript has improved in its readability. Thank you for your careful modification of your manuscript to address the reviewer comments. Your manuscript has been accepted, and I am forwarding it to the ASM Journals Department for publication. You will be notified when your proofs are ready to be viewed.
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